Novel enzymes and genes coding for the same derived from methylophilus methylotrophus

ABSTRACT

There are provided novel 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase and prephenate dehydratase/chorismate mutase and DNAs coding the enzymes derived from  Methylophilus methylotrophus.

TECHNICAL FIELD

[0001] The present invention relates to biotechnology, and morespecifically to 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase,prephenate dehydratase and genes coding for the enzymes. The genes areuseful for improvement of productivity of aromatic amino acids.

BACKGROUND ART

[0002] Conventionally, L-amino acids have been industrially produced byfermentation method utilizing microorganisms belonging to the generaBrevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces,Pseudomonas, Arthrobactor, Serratia, Penicillum and Candida. As thesemicroorganisms, strains isolated from nature or mutants of thesemicroorganisms have been used to improve the productivity. Further,there have been disclosed various recombinant DNA techniques to improveL-amino acids productivity by enhancing enzymatic activities involvingin L-amino acid-biosynthetic pathways.

[0003] Though the productivity of L-amino acids has been improved bybreeding of aforementioned microorganisms or improving productionprocesses, it is still desired to develop more inexpensive and efficientprocesses for producing L-amino acids in order to meet the expectedmarkedly increased future demand of the L-amino acids.

[0004] Conventionally, there have been known the methods for producingamino acids by fermentation using methanol as raw material which is ableto get inexpensively and massively, utilizing the bacteria belonging togenera Achromobactor and Pseudomonas (See Japanese Patent Laid-open No.45-25273), Protaminobactor (See Japanese Patent Laid-open No.49-125590), Protaminobactor and Methanomonas (See Japanese PatentLaid-open No. 5025790), Microcyclus (See Japanese Patent Laid-open No.52-18886), Methylobacillus (See Japanese Patent Laid-open No. 4-91793)and Bacillus (See Japanese Patent Laid-open No. 3-505284).

[0005] Besides, there are several enzymes that play a central role inthe biosynthetic pathway of aromatic compounds such as L-phenylalanine,L-tyrosine and L-tryptophan. The key enzyme is3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (hereafterabbreviated as “DS”). For biosynthesis of L-phenylalanine, prephenatedehydratase (hereafter abbreviated as “PD”) is also key enzyme.

[0006] However, it has not been known either gene encoding DS and PD ofa bacterium belonging to the genus Methylophilus.

Disclosure Of The Invention

[0007] An object of the present invention is to provide the genesencoding DS and PD of a bacterium belonging to the genus Methylotrophus.

[0008] To achieve the aforementioned object, the present inventorsintensively studied. As a result, they succeeded in isolating genescoding for DS and PD from Methylophilus methylotrophus using chorismatemutase-prephenate dehydratase gene (pheA)-deficient strain ofEscherichia coli, and have completed the present invention.

[0009] That is, the present invention provides:

[0010] (1) A protein as defined in the following (A) or (B):

[0011] (A) a protein which comprises the amino acid sequence depicted inSEQ ID NO: 2; or

[0012] (B) a protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 2 and which hasthe 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.

[0013] (2) A DNA coding for a protein as defined in the following (A) or(B):

[0014] (A) a protein which comprises the amino acid sequence depicted inSEQ ID NO: 2; or

[0015] (B) a protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 2 and which hasthe 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.

[0016] (3) The DNA according to (2), which is a DNA as defined in thefollowing (A) or (B):

[0017] (A) a DNA which comprises the nucleotide sequence depicted in SEQID NO: 1; or

[0018] (B) a DNA which is hybridizable with the nucleotide sequencedepicted in SEQ ID NO: 1 or the probe prepared from said sequence understringent condition and which code for a protein which has3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.

[0019] (4) The DNA according to (3), wherein the stringent condition isthe condition in which washing is performed at 60° C., and at a saltconcentration corresponding to 1×SSC and 0.1% SDS.

[0020] (5) A protein as defined in the following (C) or (D):

[0021] (C) A protein which comprises the amino acid sequence depicted inSEQ ID NO: 4; or

[0022] (D) A protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 4 and which hasat least one of the prephenate dehydratase activity or the chorismatemutase activity.

[0023] (6) A DNA coding for a protein as defined in the following (C) or(D):

[0024] (C) A protein which comprises the amino acid sequence depicted inSEQ ID NO: 4; or

[0025] (D) A protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 4 and which hasat least one of the prephenate dehydratase or the chorismate mutaseactivity.

[0026] (7) The DNA according to (6), which is a DNA as defined in thefollowing (c) or (d):

[0027] (c) A DNA which comprises the nucleotide sequence depicted in SEQID NO: 3; or

[0028] (d) A DNA which is hybridizable with the nucleotide sequencedepicted in SEQ ID NO: 3 or the probe prepared from said sequence understringent condition and which code for a protein which has at least oneof the prephenate dehydratase or chorismate mutase activity.

[0029] (8) The DNA according to (3), wherein the stringent condition isthe condition in which washing is performed at 60° C., and at a saltconcentration corresponding to 1×SSC and 0.1% SDS.

[0030] In the present invention, the term“3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity” means anactivity which catalyses a reaction to synthesize 3-deoxy-D-arabinohptulosonate-7-phosphate from phosphoenolpyruvate and D-erythrose4-phosphate. The term “prephenate dehydratase activity” means anactivity which catalyses a reaction to synthesize phenylpyruvic acidfrom prephenic acid, the term “chorismate mutase” means an activitywhich catalyses a reaction to synthesize prephenic acid from chorismicacid. It is suggested that PD described in the present invention haschorismate mutase activity as well as prephenate dehydratase activitylike other microorganism such as Escherichia coli. In the presentinvention, the term “at least one of prephenate dehydratase orchorismate mutase activity” means one or both of the properties which PDpossesses. Hereafter, in the present invention, “at least one ofprephenate dehydratase or chorismate mutase activity” may be referred toas “PD activity”.

[0031] The present invention will be explained in detail hereinafter.

[0032] The DNAs of the present invention may be obtained fromchromosomal DNA of M. methylotrophus as described below.

[0033] Chromosomal DNA of M. methylotrophus, for example, M.methylotrophus strain AS-1 is prepared. Chromosomal DNA can be obtainedfrom the cell pellet by means of, for example, a method of Saito andMiura (Biochem. Biophys. Acta., 72, 619 (1963)), or a method of K. S.Kirby (Biochem. J., 64, 405 (1956)).

[0034] Then, in order to isolate the DS or PD gene from the chromosomalDNA thus obtained, a chromosomal DNA library is prepared. At first, thechromosomal DNA is partially digested with a suitable restriction enzymeto obtain a mixture of various fragments. A wide variety of restrictionenzymes can be used if the degree of cutting is controlled by thecutting reaction time and the like. For example, Sau3AI or BamHI isallowed to react on the chromosomal DNA at a temperature not less than30° C., preferably at 37° C. at an enzyme concentration of 1-10 units/mlfor various periods of time (1 minute to 2 hours) to digest it.

[0035] Next, obtained DNA fragments are ligated with a vector DNAautonomously replicable in cells of bacteria belonging to the genusEscherichia to prepare recombinant DNA. Concretely, a restrictionenzyme, which generates the terminal nucleotide sequence complement tothat generated by the restriction enzyme Sau3AI used to cut thechromosomal DNA, for example, BamHI, is allowed to act on the vector DNAunder a condition of a temperature not less than 30° C. and an enzymeconcentration of 1-100 units/ml for not less than 1 hour, preferably for1-3 hours to completely digest it, and cut and cleave it. Next, thechromosomal DNA fragment mixture obtained as described above is mixedwith the cleaved and cut vector DNA, on which DNA ligase, preferably T4DNA ligase is allow d to act under a condition of a temperature of 4-16°C. at an enzyme concentration of 1-100 units/ml for not less than 1hour, preferably for 4-24 hours to obtain recombinant DNA.

[0036] The obtained recombinant DNA is used to transform a microorganismbelonging to the genus Escherichia, for example, such as Escherichiacoli B-7078 (pheA::Tn10(Km^(R))). Then the transformants are plated onagar plates without phenylalanine and resulted colonies are inoculatedin a liquid medium and cultivated. Plasmids are recovered from the cellsto obtain DNA fragment containing PD gene.

[0037] Whether the DNA fragment obtained as described above actuallycontains PD gene or not is confirmed by sequencing the fragment and byconfirming the determined sequence contains the sequence depicted in SEQID NO: 3.

[0038] It was proved that the cloned fragment containing PD geneobtained in Example described later also contains DS gene by the factthat the fragment complements aromatic auxotrophy of AB3257 strain(aroG365⁻, aroH367⁻, aroF363⁻, thi-1, ilvC7, argE3, his-4, proA2, xyl-5galK2, lacY1, mtl-1, strA712, tfr3, tsx-358, supE44, hsdR2,zjj-202::Tn10) which is a DS-deficient strain, and by sequencing of thefragment.

[0039] In case BamHI is used to digest chromosomal DNA of Methylophilusmethylotrophus AS-1 strain, the DS and PD gene is cloned as about 10 KbBamHI-fragment.

[0040] The DS and PD gene of other bacterium belonging to genusMethylotrophus can be isolated by the same manner as described above.Further, since nucleotide sequence of the DNA of the present inventionis clarified, the DNA can be obtained from chromosomal DNA or genomiclibrary of a bacterium belonging to genus Methylophilus by PCR(polymerase chain reaction) utilizing oligonucleotides synthesized basedon the determined sequence as a primer or hybridization utilizingoligonucleotide as described above as a probe.

[0041] It may be used normal methods which are well known to the personskilled in the art to perform preparation of genomic DNA, preparation ofgenomic DNA library, hybridization, PCR, preparation of plasmid DNA,digestion and ligation of DNA, and transformation. These are describedby Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning ALaboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press,(1989).

[0042] A nucleotide sequence of DS gene obtained as described above isillustrated in SEQ ID NO: 1 in Sequence Listing. Further, an amino acidsequence of a protein which may be encoded by nucleotide sequence isillustrated in SEQ ID NO: 2.

[0043] A nucleotide sequence of PD gene obtained as described above isillustrated in SEQ ID NO: 3 in Sequence Listing. Further, an amino acidsequence of a protein which may be encoded by the nucleotide sequence isillustrated in SEQ ID NO: 4.

[0044] The DNA of the present invention may code for DS or PD includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids at one or a plurality of positions, provided thatthe activity of DS or PD encoded thereby is not deteriorated. The numberof “several” amino acids differs depending on the position or the typeof amino acid residues in the three-dimensional structure of theprotein. This is because of the following reason. That is, some aminoacids such as isoleucine and valine are amino acids having high homologyto one another. The difference in such an amino acid does not greatlyaffect the three-dimensional structure of the protein. Therefore, theprotein encoded by the DNA of the present invention may be one which hashomology of not less than 35 to 50%, preferably 50 to 70% with respectto the entire amino acid residues for constituting DS or PD, and whichhas the DS and PD activity. More appropriately, the number of “several”amino acids is 2 to 30, preferably 2 to 20, and more preferably 2 to 10.

[0045] DNA, which codes for the substantially same protein as DS or PDas described above, is obtained, for example, by modifying thenucleotide sequence, for example, by means of the site-directedmutagenesis method so that one or more amino acid residues at aspecified site involve substitution, deletion, insertion, addition, orinversion. DNA modified as described above may be obtained by theconventionally known mutation treatment. The mutation treatment includesa method for treating DNA coding for DS and PD in vitro, for example,with hydroxylamine, and a method for treating a microorganism, forexample, a bacterium belonging to the genus Escherichia harboring DNAcoding for DS and PD with ultraviolet irradiation or a mutating agentsuch as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acidusually used for the mutation treatment.

[0046] The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, on the basis of the individualdifference or the difference in species or genus of the microorganismharboring DS and PD.

[0047] The DNA, which codes for the substantially same protein as DS andPD, is obtained by expressing DNA having mutation as described above inan appropriate cell, and investigating the DS and PD activity of anexpressed product. The DNA, which codes for the substantially sameprotein as DS and PD, is also obtained by isolating DNA that ishybridizable with DNA having, for example, a nucleotide sequencedepicted in SEQ ID NO: 1 in Sequence Listing under a stringentcondition, and which codes for a protein having the DS and PD activity,from DNA coding for DS and PD having mutation or from a cell harboringit. The “stringent condition” referred to herein is a condition underwhich so-called specific hybrid is formed, and non-specific hybrid isnot formed. It is difficult to clearly express this condition by usingany numerical value. However, for example, the stringent conditionincludes a condition under which DNA's having high homology, forexample, DNA's having homology of not less than 50% are hybridized witheach other, and DNA's having homology lower than the above are nothybridized with each other. Alternatively, the stringent condition isexemplified by a condition under which DNA's are hybridized with eachother at a salt concentration corresponding to an ordinary condition ofwashing in Southern hybridization, i.e., 60° C., 1×SSC, 0.1% SDS,preferably 0.1×SSC, 0.1% SDS.

[0048] The gene, which is hybridizable under the condition as describedabove, includes those having a stop codon generated in a coding regionof the gene, and those having no activity due to mutation of activecenter. However, such mutants can be easily removed by ligating the genewith a commercially available activity expression vector, and measuringthe DS and PD activity in accordance with the method as described above.

[0049] The host to be expressed DS or PD gene are exemplified by, forexample, bacterium belonging to the genus Escherichia such asEscherichia coli, Cornyneform bacterium such as Brevibacteriumlactofermentum, bacterium belonging to the genus Methylophilus such asMethylophilus methylotrophus, other various eukaryotes such asSaccharomyces cerevisiae, animal cells and plant cells, preferablyprokaryote, especially E. coli Coryneform bacterium and M.methylotrophus.

[0050] The vector to be introduced DS or PD gene into E. coli includes,for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010,pMW119, pMW118, pMW219 and pMW218. Phage DNA vectors may be also used.

[0051] The vector to be to be used for introducing DS or PD gene intoCoryneform bacterium includes, for example, pAM330 (see Japanese PatentLaid-open No. 58-67699), pHM1519 (see Japanese Patent Laid-open No.58-77895), pAJ655, pAJ611 and pAJ1844 (see Japanese Patent Laid-open No.58-192900), pCG1 (see Japanese Patent Laid-open No. 57-134500), pCG2(see Japanese Patent Laid-open No. 58-35197), pCG4 and pCG11 (seeJapanese Patent Laid-open No. 57-183799), pHK4 (see Japanese PatentLaid-open No. 5-7491).

[0052] Introduction of DS and PD gene may be performed by transformingthe host as described above with a recombinant vector obtained byconnecting DS or PD gene to the vector as described above. The DS or PDgene may be incorporated into the genome of the host in accordance withthe method based on the use of transduction, transposon (Berg, D. E. andBerg, C. M., Bio/Technol., 1, 417 (1983)), Mu phage (Japanese Laid-OpenPatent Publication No. 2-109985), or homologous recombination(Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).

[0053] DS and PD may be produced by cultivating the cell in which DS orPD gene is introduced in accordance with the method as described above,producing and accumulating DS or PD in the medium, collecting from theculture. The medium used for cultivation may be selected appropriatelyto the host used therein.

[0054] DS or PD produced by the method as described above, if necessary,it may be purified from cell extract or medium by normal method ofpurification of enzymes such as ion exchange chromatography, gelfiltration chromatography, absorption chromatography, solventprecipitation and the like.

[0055] Microorganism having higher activity of DS or PD than that ofwild type may be constructed by using the DNA of the present invention.It can be performed by transforming microorganism with the vectorcontaining DS or PD gene as an expressible form.

[0056] Bacterium to be used for the present invention includes, forexample, Methylophilus methylotrophus AS1 (NCIMB10515) or the like. Itis possible to obtain Methylophilus methylotrophus AS1 (NCIMB10515) fromNational Collections of Industrial and Marin Bacteria, NCIMB Lts., TorryRes arch Station 135, Abbey Road, Aberdeen AB9 8DG, United Kingdom.

[0057] In order to improve the productivity of aromatic amino acids,especially for L-phenylalanine, it is useful to amplification of genesencoding the DS and PD enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 shows the construction of plasmids pPD1 and pPD2 having DSand PD genes, and

[0059]FIG. 2 shows the complementation analysis of the plasmids,obtained after the deletion of M. methylotrophus DNA fragment, carryingPD and DS genes.

BEST MODE FOR CARRYING OUT THE INVENTION

[0060] The 10 kbp BamHI DNA fragment carrying the M. methylotrophusprephenate dehydratase and DAHP-synthase genes, was cloned on a low copyvector pMW119 (Ap^(R)) in shotgun experiments by complementation (FIG.1). In this experiment the chromosomal DNA of the M. methylotrophus AS-1was digested with BamHI and the resulting DNA fragments were ligatedwith BamHI digestion product of plasmid pMW119 using T4 DNA ligase. Theligation product was used to transform E. coli B-7078 strain(pheA::Tn10(Km^(R))). Among the clones resistant to ampicillin, thestrains in which phenylalanine auxotrophy disappeared were selected, andthe recombinant plasmids were recovered from the selected strains. Oneof the resulted plasmids was named as pPD1. The plasmid with theopposite orientation of the cloned fragment to that of pPD1 was named aspPD2.

[0061] The plasmids pPD1 and pPD2 complemented to the prototrophy notonly the pheA⁻ mutation of E. coli B-7078 strain, but also the DS-minusE. coli AB3257 strain (aroG⁻, aroH⁻, aroF⁻). Thus, the both plasmidspPD1 and pPD2 were supposed to bear the genes encoding PD enzyme and DSenzyme in the same cloned DNA fragment.

[0062] The deletion derivatives of pPD1 and pPD2 were constructed (FIG.2). The deletions were performed in vitro by digesting the plasmid DNAwith different restriction enzymes and ligating resulting DNA fragments.The ligation mixtures were used to transform the PD-minus strain E. coliB-7078 (pheA::Tn10 (kan)) to a Phe⁺ prototrophy. The isolated plasmidswere mapped and tested in the ability to complement the DS-minus mutantE. coli AB3257 (aroG⁻, aroH⁻, aroF⁻) to Aro⁺ prototrophy. Theconstructed deletion derivatives were varied in structure andcomplementation ability. The deletion derivatives carrying only one geneencoding PD enzyme were found. These deletion derivatives lost anability to complement DS-minus mutant E. coli AB3257 (aroG⁻, aroH⁻,aroF⁻) to prototrophy. Thus, the cloned M. methylotrophus DNA fragmentin pPD1 or pPD2 was supposed to carry two different genes encoding DSand PD enzymes, respectively.

[0063] The nucleotide sequences of the M. methylotrophus two genesencoding DS and PD enzymes were determined. The nucleotide sequence ofthe DS gene and the amino acid sequence coded by the nucleotide sequenceare shown in SEQ ID NO: 1. The nucleotide sequence of the PD gene andthe amino acid sequence coded by the nucleotide sequence are shown inSEQ ID NO: 3.

[0064] The nucleotide sequences of M. methylotrophus genes encoding DSand PD were analyzed and characterized by using the computer programs.The DS and PD gene sequences after translation showed a significantamino acid sequence similarity with the same function proteins of manyother microorganisms (Table 1, 2).

INDUSTRIAL APPLICABILITY

[0065] The present invention provides3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, prephenatedehydratase and genes coding for the enzymes. The genes are useful forimprovement of productivity of aromatic amino acids TABLE 1 Thealignment of M. methylotrophus DS enzyme protein sequence with thesimilar sequences of other microorganisms Homology Sequence (%) withaccession M.m. DS Microorganism number Gene Enzyme protein Echerichiacoli P00886 aroG 3-deoxy-D-arabino- 60% heptulosonate 7- phosphatesynthase Haemophilus P44303 aroG 3-deoxy-D-arabino- 56% influenzaeheptulosonate 7- phosphate synthase Echerichia coli P00887 aroH3-deoxy-D-arabino- 54% heptulosonate 7- phosphate synthase Schizosaccha-Q09755 aroF 3-deoxy-D-arabino- 52% romyces pombe heptulosonate 7-phosphate synthase Erwinia herbicola O54459 aroH 3-deoxy-D-arabino- 52%heptulosonate 7- phosphate synthase Saccharomyces P32449 aroG3-deoxy-D-arabino- 54% cerevisiae heptulosonate 7- phosphate synthaseEcherichia coli P00888 aroF 3-deoxy-D-arabino- 49% heptulosonate 7-phosphate synthase Candida albicans P79023 aroG 3-deoxy-D-arabino- 51%heptulosonate 7- phosphate synthase Salmonella P21307 aroF 3-deoxy-D-arabino- 49% typhimurium heptulosonate 7- phosphate synthase BuchneraP46245 aroH 3-deoxy-D-arabino- 47% aphidicola heptulosonate 7- phosphatesynthase Saccharomyces P14843 aroF 3-deoxy-D-arabino- 49% cerevisiaeheptulosonate 7- phosphate synthase Corynebacterium P35170 aroG3-deoxy-D-arabino- 48% glutamicum heptulosonate 7- phosphate synthaseCandida albicans P34725 aroF 3-deoxy-D- 50% arabino-heptulosonate7-phosphate synthase Erwinia herbicola Q02285 aroF 3-deoxy-D- 53%arabino-heptulosonate 7-phosphate synthase Amycolatopsis Q44093 aroG3-deoxy-D- 52% methanolica arabino-heptulosonate 7-phosphate synthase

[0066] TABLE 2 The alignment of M. methylotrophus PD enzyme proteinsequence with the similar sequences of other microorganisms HomologySequence (%) with accession M.m. PD Microorganism number Gene Enzymeprotein Neisseria Q9ZHY3 pheA Chorismate mutase; 54% gonorrhoeaePrephenate dehydratase Pseudomonas P27603 pheA Chorismate mutase; 47%stutzeri Prephenate dehydratase Aquifex O67085 pheA Chorismate mutase;47% aeolicus Prephenate dehydratase Erwinia Q02286 pheA Chorismatemutase; 37% herbicola Prephenate dehydratase Echerichia coli P07022 pheAChorismate mutase; 36% Prephenate dehydratase Haemophilus P43900 pheAChorismate mutase; 34% influenzae Prephenate dehydratase

[0067]

1 4 1 1083 DNA Methylophilus methylotrophus CDS (1)..(1080) 1 atg actgca tac gaa aaa tta gcc acc gat gat gtg cgc gtg ctt gaa 48 Met Thr AlaTyr Glu Lys Leu Ala Thr Asp Asp Val Arg Val Leu Glu 1 5 10 15 atc aagccg ctg gta aag ccc gcg gag cta ttg tct cgc ctg cag gaa 96 Ile Lys ProLeu Val Lys Pro Ala Glu Leu Leu Ser Arg Leu Gln Glu 20 25 30 agt aca gtcagt acc caa aac atc ctt aaa acg cgg tca gcg att cat 144 Ser Thr Val SerThr Gln Asn Ile Leu Lys Thr Arg Ser Ala Ile His 35 40 45 cat att ctc catcag ggc gac gac cgg ttg ctg gtg att gtt ggc cct 192 His Ile Leu His GlnGly Asp Asp Arg Leu Leu Val Ile Val Gly Pro 50 55 60 tgt tcc atc cat gacacg gaa gct ggc atg gag tac gcg cga cgc ctg 240 Cys Ser Ile His Asp ThrGlu Ala Gly Met Glu Tyr Ala Arg Arg Leu 65 70 75 80 ctc gat gtg cgt cagcga ctg ggt ggc gaa ttg ctc att gtc atg cgc 288 Leu Asp Val Arg Gln ArgLeu Gly Gly Glu Leu Leu Ile Val Met Arg 85 90 95 gtc tat ttt gag aaa ccccgt acc acg gta ggg tgg aaa ggc ctg atc 336 Val Tyr Phe Glu Lys Pro ArgThr Thr Val Gly Trp Lys Gly Leu Ile 100 105 110 aac gac ccg cat ctg gatggg act tat gat atc aat ctt gga ttg gag 384 Asn Asp Pro His Leu Asp GlyThr Tyr Asp Ile Asn Leu Gly Leu Glu 115 120 125 aag gcc cgc cgt ttc ctgctg gat gtg aat gaa att ggc atg cct gca 432 Lys Ala Arg Arg Phe Leu LeuAsp Val Asn Glu Ile Gly Met Pro Ala 130 135 140 gcc aca gaa ttc ctc gatgtg gtc tcc ccg caa tat act gct gac ctg 480 Ala Thr Glu Phe Leu Asp ValVal Ser Pro Gln Tyr Thr Ala Asp Leu 145 150 155 160 gtc agc tgg gga gctatt ggc gct cgg acg aca gag tct cag att cac 528 Val Ser Trp Gly Ala IleGly Ala Arg Thr Thr Glu Ser Gln Ile His 165 170 175 cgc gaa ttg gcc tctggc ctg tct tgt ccg gtt ggc ttt aaa aat ggg 576 Arg Glu Leu Ala Ser GlyLeu Ser Cys Pro Val Gly Phe Lys Asn Gly 180 185 190 acc gat ggc ggc gtcaaa gtt gcc att gat gcg att aag gca gca gcc 624 Thr Asp Gly Gly Val LysVal Ala Ile Asp Ala Ile Lys Ala Ala Ala 195 200 205 agt ccg cat cac tttttg tcc gtg acc aaa gaa ggc gaa tcc gct att 672 Ser Pro His His Phe LeuSer Val Thr Lys Glu Gly Glu Ser Ala Ile 210 215 220 ttt gcc acc aag ggtaat gaa gac tgc cat gtg att tta cgt ggc ggt 720 Phe Ala Thr Lys Gly AsnGlu Asp Cys His Val Ile Leu Arg Gly Gly 225 230 235 240 aaa gcg ccg aacttt gat gcg cct agt gtg gca gca gta tgc gac caa 768 Lys Ala Pro Asn PheAsp Ala Pro Ser Val Ala Ala Val Cys Asp Gln 245 250 255 ttg gca gac gctggc ctg gca ccg gta ttg atg gtg gat tgc agt cat 816 Leu Ala Asp Ala GlyLeu Ala Pro Val Leu Met Val Asp Cys Ser His 260 265 270 ggc aat agc cagaag caa tat aaa aac caa att tcg gtg gtg aat gat 864 Gly Asn Ser Gln LysGln Tyr Lys Asn Gln Ile Ser Val Val Asn Asp 275 280 285 gtg gct agc caaata gcg ggt gga gat gct cgc ata atc ggg atc atg 912 Val Ala Ser Gln IleAla Gly Gly Asp Ala Arg Ile Ile Gly Ile Met 290 295 300 cta gag tcg catttg aac gaa ggg cga cag gat cat tcg cca ggc tgc 960 Leu Glu Ser His LeuAsn Glu Gly Arg Gln Asp His Ser Pro Gly Cys 305 310 315 320 agc ctt aattat ggg caa tcc atc acc gat gcc tgt ttg gga tgg gag 1008 Ser Leu Asn TyrGly Gln Ser Ile Thr Asp Ala Cys Leu Gly Trp Glu 325 330 335 gac tca gtggct gtg ctg gaa acg ctg gct gct gca gtc aag gcc cgc 1056 Asp Ser Val AlaVal Leu Glu Thr Leu Ala Ala Ala Val Lys Ala Arg 340 345 350 cgt gac aagcat gcc gct gct gaa taa 1083 Arg Asp Lys His Ala Ala Ala Glu 355 360 2360 PRT Methylophilus methylotrophus 2 Met Thr Ala Tyr Glu Lys Leu AlaThr Asp Asp Val Arg Val Leu Glu 1 5 10 15 Ile Lys Pro Leu Val Lys ProAla Glu Leu Leu Ser Arg Leu Gln Glu 20 25 30 Ser Thr Val Ser Thr Gln AsnIle Leu Lys Thr Arg Ser Ala Ile His 35 40 45 His Ile Leu His Gln Gly AspAsp Arg Leu Leu Val Ile Val Gly Pro 50 55 60 Cys Ser Ile His Asp Thr GluAla Gly Met Glu Tyr Ala Arg Arg Leu 65 70 75 80 Leu Asp Val Arg Gln ArgLeu Gly Gly Glu Leu Leu Ile Val Met Arg 85 90 95 Val Tyr Phe Glu Lys ProArg Thr Thr Val Gly Trp Lys Gly Leu Ile 100 105 110 Asn Asp Pro His LeuAsp Gly Thr Tyr Asp Ile Asn Leu Gly Leu Glu 115 120 125 Lys Ala Arg ArgPhe Leu Leu Asp Val Asn Glu Ile Gly Met Pro Ala 130 135 140 Ala Thr GluPhe Leu Asp Val Val Ser Pro Gln Tyr Thr Ala Asp Leu 145 150 155 160 ValSer Trp Gly Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Ile His 165 170 175Arg Glu Leu Ala Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly 180 185190 Thr Asp Gly Gly Val Lys Val Ala Ile Asp Ala Ile Lys Ala Ala Ala 195200 205 Ser Pro His His Phe Leu Ser Val Thr Lys Glu Gly Glu Ser Ala Ile210 215 220 Phe Ala Thr Lys Gly Asn Glu Asp Cys His Val Ile Leu Arg GlyGly 225 230 235 240 Lys Ala Pro Asn Phe Asp Ala Pro Ser Val Ala Ala ValCys Asp Gln 245 250 255 Leu Ala Asp Ala Gly Leu Ala Pro Val Leu Met ValAsp Cys Ser His 260 265 270 Gly Asn Ser Gln Lys Gln Tyr Lys Asn Gln IleSer Val Val Asn Asp 275 280 285 Val Ala Ser Gln Ile Ala Gly Gly Asp AlaArg Ile Ile Gly Ile Met 290 295 300 Leu Glu Ser His Leu Asn Glu Gly ArgGln Asp His Ser Pro Gly Cys 305 310 315 320 Ser Leu Asn Tyr Gly Gln SerIle Thr Asp Ala Cys Leu Gly Trp Glu 325 330 335 Asp Ser Val Ala Val LeuGlu Thr Leu Ala Ala Ala Val Lys Ala Arg 340 345 350 Arg Asp Lys His AlaAla Ala Glu 355 360 3 1083 DNA Methylophilus methylotrophus CDS(1)..(1080) 3 atg tct gat tta tta aaa caa ttt aga gat aag att gac gcgatt gat 48 Met Ser Asp Leu Leu Lys Gln Phe Arg Asp Lys Ile Asp Ala IleAsp 1 5 10 15 gcg cag att cta gcg ctc gtc aat gag cgt gcc aag ctg gcacgt gaa 96 Ala Gln Ile Leu Ala Leu Val Asn Glu Arg Ala Lys Leu Ala ArgGlu 20 25 30 atc ggc cat tta aag gat gat ggt gtg att tac cgt cct gag cgtgaa 144 Ile Gly His Leu Lys Asp Asp Gly Val Ile Tyr Arg Pro Glu Arg Glu35 40 45 gcg caa att atc cgt cgc ttg caa gca gaa aat gaa ggg ccg ctg tca192 Ala Gln Ile Ile Arg Arg Leu Gln Ala Glu Asn Glu Gly Pro Leu Ser 5055 60 ccg gag gcc gtc agc cat att ttc cgt gcg gtc atg tcc aat tgt cgc240 Pro Glu Ala Val Ser His Ile Phe Arg Ala Val Met Ser Asn Cys Arg 6570 75 80 gct ttg gaa aaa gaa ctt gcg att gcc ttt ttg ggc cca ctg ggc acc288 Ala Leu Glu Lys Glu Leu Ala Ile Ala Phe Leu Gly Pro Leu Gly Thr 8590 95 tac agt gaa gaa gcc gca ctc aag cag ttt ggt gaa ggc cgc cag gca336 Tyr Ser Glu Glu Ala Ala Leu Lys Gln Phe Gly Glu Gly Arg Gln Ala 100105 110 gtc gtc tgc ggc agt att gat gaa gtt ttt cgt acg gtg gaa gct ggc384 Val Val Cys Gly Ser Ile Asp Glu Val Phe Arg Thr Val Glu Ala Gly 115120 125 cag gcg gat tac ggc gtt gtc cct gta gaa aac tca acc gaa ggt gcg432 Gln Ala Asp Tyr Gly Val Val Pro Val Glu Asn Ser Thr Glu Gly Ala 130135 140 gtg gga att acg ctg gac tta tta ctg ggt agt gcg ctg caa gtg gta480 Val Gly Ile Thr Leu Asp Leu Leu Leu Gly Ser Ala Leu Gln Val Val 145150 155 160 ggc gag gtg act tta cca gta cat cac tgc ttg cta tcg gcc cagcag 528 Gly Glu Val Thr Leu Pro Val His His Cys Leu Leu Ser Ala Gln Gln165 170 175 gat ttg caa cag atc acg cat gtg ttc tcg cac gca cag tct ttgtcg 576 Asp Leu Gln Gln Ile Thr His Val Phe Ser His Ala Gln Ser Leu Ser180 185 190 caa tgt cat gaa tgg cta aat aaa gtg tta ccg agt gca caa cgagaa 624 Gln Cys His Glu Trp Leu Asn Lys Val Leu Pro Ser Ala Gln Arg Glu195 200 205 gct gtg acc agc aac gcg cgt gct gca caa atg att cat gag ctagtc 672 Ala Val Thr Ser Asn Ala Arg Ala Ala Gln Met Ile His Glu Leu Val210 215 220 gcc acc caa ggc acg ttt gcg gct gcg att gcc agc aaa cgt gcggct 720 Ala Thr Gln Gly Thr Phe Ala Ala Ala Ile Ala Ser Lys Arg Ala Ala225 230 235 240 gaa ttg ttt gac ttg aat ata ctc gcc gaa aat atc gaa gatgat ccg 768 Glu Leu Phe Asp Leu Asn Ile Leu Ala Glu Asn Ile Glu Asp AspPro 245 250 255 aaa aat acc acg cgc ttt ctg gtg ttg ggt aat cac ggc gtcgca cct 816 Lys Asn Thr Thr Arg Phe Leu Val Leu Gly Asn His Gly Val AlaPro 260 265 270 tct ggt cag gat aaa acc tcg ttg gtg atg agt gct cac aacaag cca 864 Ser Gly Gln Asp Lys Thr Ser Leu Val Met Ser Ala His Asn LysPro 275 280 285 ggc gcg gtg ttg caa ttg ctg gaa cca ttg tca cgc cat ggcgtg agt 912 Gly Ala Val Leu Gln Leu Leu Glu Pro Leu Ser Arg His Gly ValSer 290 295 300 atg acc aag ctg gaa tcg cgt cca tca cgt caa aat cta tggaac tac 960 Met Thr Lys Leu Glu Ser Arg Pro Ser Arg Gln Asn Leu Trp AsnTyr 305 310 315 320 gta ttt ttt gtt gac att gaa ggt cat caa cag cag ccctcg gta caa 1008 Val Phe Phe Val Asp Ile Glu Gly His Gln Gln Gln Pro SerVal Gln 325 330 335 gct gcg ctg aaa gaa ctg gct gag cgc gcg act ttc cttaaa gtg ttg 1056 Ala Ala Leu Lys Glu Leu Ala Glu Arg Ala Thr Phe Leu LysVal Leu 340 345 350 ggc tca tac cca acc gct att att taa 1083 Gly Ser TyrPro Thr Ala Ile Ile 355 360 4 360 PRT Methylophilus methylotrophus 4 MetSer Asp Leu Leu Lys Gln Phe Arg Asp Lys Ile Asp Ala Ile Asp 1 5 10 15Ala Gln Ile Leu Ala Leu Val Asn Glu Arg Ala Lys Leu Ala Arg Glu 20 25 30Ile Gly His Leu Lys Asp Asp Gly Val Ile Tyr Arg Pro Glu Arg Glu 35 40 45Ala Gln Ile Ile Arg Arg Leu Gln Ala Glu Asn Glu Gly Pro Leu Ser 50 55 60Pro Glu Ala Val Ser His Ile Phe Arg Ala Val Met Ser Asn Cys Arg 65 70 7580 Ala Leu Glu Lys Glu Leu Ala Ile Ala Phe Leu Gly Pro Leu Gly Thr 85 9095 Tyr Ser Glu Glu Ala Ala Leu Lys Gln Phe Gly Glu Gly Arg Gln Ala 100105 110 Val Val Cys Gly Ser Ile Asp Glu Val Phe Arg Thr Val Glu Ala Gly115 120 125 Gln Ala Asp Tyr Gly Val Val Pro Val Glu Asn Ser Thr Glu GlyAla 130 135 140 Val Gly Ile Thr Leu Asp Leu Leu Leu Gly Ser Ala Leu GlnVal Val 145 150 155 160 Gly Glu Val Thr Leu Pro Val His His Cys Leu LeuSer Ala Gln Gln 165 170 175 Asp Leu Gln Gln Ile Thr His Val Phe Ser HisAla Gln Ser Leu Ser 180 185 190 Gln Cys His Glu Trp Leu Asn Lys Val LeuPro Ser Ala Gln Arg Glu 195 200 205 Ala Val Thr Ser Asn Ala Arg Ala AlaGln Met Ile His Glu Leu Val 210 215 220 Ala Thr Gln Gly Thr Phe Ala AlaAla Ile Ala Ser Lys Arg Ala Ala 225 230 235 240 Glu Leu Phe Asp Leu AsnIle Leu Ala Glu Asn Ile Glu Asp Asp Pro 245 250 255 Lys Asn Thr Thr ArgPhe Leu Val Leu Gly Asn His Gly Val Ala Pro 260 265 270 Ser Gly Gln AspLys Thr Ser Leu Val Met Ser Ala His Asn Lys Pro 275 280 285 Gly Ala ValLeu Gln Leu Leu Glu Pro Leu Ser Arg His Gly Val Ser 290 295 300 Met ThrLys Leu Glu Ser Arg Pro Ser Arg Gln Asn Leu Trp Asn Tyr 305 310 315 320Val Phe Phe Val Asp Ile Glu Gly His Gln Gln Gln Pro Ser Val Gln 325 330335 Ala Ala Leu Lys Glu Leu Ala Glu Arg Ala Thr Phe Leu Lys Val Leu 340345 350 Gly Ser Tyr Pro Thr Ala Ile Ile 355 360

1. A protein as defined in the following (A) or (b): (A) a protein whichcomprises the amino acid sequence depicted in SEQ ID NO: 2; or (B) aprotein which comprises the amino acid sequence including deletion,substitution, insertion or addition of one or several amino acids in theamino acid sequence depicted in SEQ ID NO: 2 and which has the3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.
 2. A DNAcoding for a protein as defined in the following (A) or (B): (A) aprotein which comprises the amino acid sequence depicted in SEQ ID NO:2; or (B) a protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 2 and which hasthe 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.
 3. TheDNA according to claim 2, which is a DNA as defined in the following (A)or (B): (A) a DNA which comprises the nucleotide sequence depicted inSEQ ID NO: 1; or (B) a DNA which is hybridizable with the nucleotidesequence depicted in SEQ ID NO: 1 or the probe prepared from saidsequence under stringent condition and which code for a protein whichhas 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase activity.
 4. TheDNA according to claim 3, wherein the stringent condition is thecondition in which washing is performed at 60° C., and at a saltconcentration corresponding to 1×SSC and 0.1% SDS.
 5. A protein asdefined in the following (C) or (D): (C) A protein which comprises theamino acid sequence depicted in SEQ ID NO: 4; or (D) A protein whichcomprises the amino acid sequence including deletion, substitution,insertion or addition of one or several amino acids in the amino acidsequence depicted in SEQ ID NO: 4 and which has at least one of theprephenate dehydratase activity or the chorismate mutase activity.
 6. ADNA coding for a protein as defined in the following (C) or (D): (C) Aprotein which comprises the amino acid sequence depicted in SEQ ID NO:4; or (D) A protein which comprises the amino acid sequence includingdeletion, substitution, insertion or addition of one or several aminoacids in the amino acid sequence depicted in SEQ ID NO: 4 and which hasat least one of the prephenate dehydratase or the chorismate mutaseactivity.
 7. The DNA according to claim 6, which is a DNA as defined inthe following (c) or (d): (c) A DNA which comprises the nucleotidesequence depicted in SEQ ID NO: 3; or (d) A DNA which is hybridizablewith the nucleotide sequence depicted in SEQ ID NO: 3 or the probeprepared from said sequence under stringent condition and which code fora protein which has at least one of the prephenate dehydratase orchorismate mutase activity.
 8. The DNA according to claim 7, wherein thestringent condition is the condition in which washing is performed at60° C., and at a salt concentration corresponding to 1×SSC and 0.1% SDS.